Enzyme with acetyl esterase activity

ABSTRACT

An isolated and purified acetyl esterase with activity towards acetylated xylan and acetylated mannan obtained from Aspergillus aculeatus and having amino acid sequence that comprises Seq ID No 1. An enzyme can be used for the modification of plant cell wall components.

FIELD OF INVENTION

The present invention relates to enzymes with acetyl esterase activity,a method of producing the enzymes, and an enzyme preparation containingone or more of the enzymes.

BACKGROUND OF THE INVENTION

Many polysaccharides can exist in acetylated forms in various biologicalplant materials (mainly in xylan, mannan and pectin polymers) 1!. Thebiological significance of the acetyl groups is not fully understood. Itis known that the acetyl group often protects the polysaccharide fromdegradation by hydrolytic enzymes. Hence, deacetylation of thesepolysaccharides is necessary in order to achieve partial or completeenzymatic breakdown of the acetylated polysaccharide 2-4!.

Accordingly, it is contemplated 2,3! that acetyl esterases are importantenzymes for the food industry, primarily in fruit and vegetableprocessing such as fruit juice production, wine making or pectinextraction, where their ability to modify acetylated polysaccharides toa readily degradable form may be utilised.

It is known that many fungi contain enzymes capable of deacetylatingacetylated polysaccharides, which enzymes are commonly designated acetylesterases. Some fungal acetyl esterases have been purified 5-9!.However, the study of these enzymes have been hampered by the lack ofwell-characterized homogeneous substrates, and by the difficult and timeconsuming assays for measuring acetate release. Any industrial use ofthese enzymes has not been described.

WO 92/19728 describes a rhamnogalacturonan acetyl esterase isolated fromthe fungal species Aspergillus aculeatus. This enzyme is specific foracetylated galacturonic acid residues in hairy regions of pectin. EP 507369 discloses a DNA sequence encoding an acetyl xylan esterase isolatedfrom Aspergillus niger.

For many purposes, it would be desirable to provide acetyl esterases ina form essentially free from other components. In this way, it would bepossible to produce enzyme preparations adapted to specific purposes,such preparations either containing a single acetyl esterase orarbitrary combinations thereof, and optionally containing otherpolysaccharide degrading enzymes. To serve this end, it is convenient toprovide single-component acetyl esterases by recombinant DNA techniques.

SUMMARY OF THE INVENTION

It has now surprisingly been found that the fungal species A. aculeatus,in addition to the above mentioned rhamnogalacturonan acetylesterase,produces a number of novel acetyl esterases with interesting enzymaticactivities. Despite the fact that these enzymes are produced in very lowamounts (constituting less than 0.1% of the total enzyme production),the present inventors have succeeded in purifying and characterizing thenovel enzymes.

Accordingly, the present invention relates to novel enzymes havingacetyl esterase activity and in particular to single-component acetylesterases.

More specifically, in a first aspect the present invention relates to anenzyme with acetyl esterase activity, which enzyme comprises the aminoacid sequence shown in SEQ ID No. 1, in which x designates any aminoacid residue.

In the present context the term "with acetyl esterase activity" is usedto define a group of enzymes, the members of which have the commoncharacteristic of being capable of cleaving the acetyl esterasesubstrate p-nitrophenol-acetate (PNP-acetate) by the procedure given inthe Materials and Methods section below. Furthermore, they may in somecases be able to act on other acetylated non-saccharide substrates.These enzymes are commonly termed acetyl esterases. It will beunderstood that the natural substrate for each of the acetyl esterasesdisclosed herein may vary between different acetylated polysaccharides,as exemplified by acetylated mannans, acetylated xylans, acetylatedrhamnogalacturonans and acetylated pectins.

In the course of the research leading to the present invention is it wassurprisingly found that different acetyl esterases isolated from A.aculeatus comprise the partial amino acid sequence shown in SEQ IDNo. 1. The presence of this sequence may be a characteristic feature ofacetyl esterases, in particular of acetyl esterases produced by A.aculeatus or by other related organisms. As stated above, x may be anyamino acid sequence which in the present context is intended to beunderstood to comprise alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine and valine.

In a second aspect the present invention relates to an enzyme withacetyl esterase activity, which enzyme is active towards acetylatedxylan as well as acetylated mannan. As far as the present inventors areaware there has been no previous disclosure of an enzyme having activitytowards both of these substrates. Thus, acetyl esterases are known to bevery specific as they only deacetylate one type of acetylatedcarbohydrate. The acetyl esterase according to this aspect of theinvention is advantageous in that it can be used for more than one typeof substrate.

In the present context the terms "activity towards acetylated xylan" and"activity towards acetylated mannan" are intended to indicate that theenzyme is capable of hydrolyzing the ester linkage found between acetylgroups and xylan and mannan, respectively. A suitable assay fordetermining activity towards the two substrates are given in theMaterials and Methods section below.

In a further aspect the invention relates to an enzyme with acetylesterase activity, which enzyme is immunologically reactive with anantibody raised against a purified acetyl esterase derived fromAspergillus aculeatus, CBS 101.43 and having a molecular weight of about44 or 35 kDa.

The A. aculeatus acetyl esterase having a molecular weight of about 44kDa or about 35 kDa is as defined herein. It will be understood that theexact value obtained for the molecular weight of a given enzyme willdepend on the method used for its determination. Thus, variations in theactual value determined may occur, even when only a slightly differentMw-determination method is used and/or when the Mw-determination iscarried out by two different persons. Thus, the molecular weights ofenzymes given herein should be evaluated on this basis and interpretedrather as a range around the actual value stated, than an exact value.If the molecular weight of the enzyme is determined by any othersuitable method known in the art (e.g. mass spectromety, gel filtrationor sedimentation), than that actually used in the present context (whichis further described in the Materials and Methods section below) one maythus expect a different Mw to be obtained than that actually stated inthe present application.

In the present context, the term "derived from" is intended not only toindicate an acetyl esterase produced by strain CBS 101.43, but also anacetyl esterase encoded by a DNA sequence isolated from strain CBS101.43 and produced in a host organism transformed with said DNAsequence.

In a still further aspect the invention relates to an enzyme with acetylesterase activity, which enzyme is encoded by a DNA sequence comprisingthe DNA sequence shown in SEQ ID No. 4.

It will be understood that also an enzyme encoded by a DNA sequencecomprising an analogue of the DNA sequence shown in SEQ ID No. 4 is tobe considered within the present invention.

In the present context the term "analogue" is understood to include anyDNA sequence which encodes an enzyme with acetyl esterase activity andwhich is at least 70% homologous to the DNA sequence shown in SEQ ID No.4, including a partial sequence of the DNA sequence shown in SEQ ID No.4. The analogous DNA sequence may be a DNA sequence which hybridizes tothe same probe as the DNA coding for the acetyl esterase under thefollowing conditions: presoaking in 5×SSC and prehybridizing for 1 h at˜40° C. in a solution of 5×SSC, 5×Denhardt's solution, 50 mM sodiumphosphate, pH 6.8, and 50 μg of denatured sonicated calf thymus DNA,followed by hybridization in the same solution supplemented with 50 μCi32-P-dCTP labelled probe for 18 h at ˜40° C. followed by washing threetimes in 2×SSC, 0.2% SDS at 40° C. for 30 minutes. The analogous DNAsequence is preferably at least 80% such as at least 90% homologous tothe sequence shown in SEQ ID No. 4, preferably at least 95% homologousto said sequence.

The analogous DNA sequence may, e.g., be isolated from another organismor may be one prepared on the basis of the amino acid sequence shown inany of SEQ ID Nos. 1-3, such as by introduction of nucleotidesubstitutions which do not give rise to another amino acid sequence ofthe acetyl esterase but which correspond to the codon usage of the hostorganism into which the DNA construct is introduced or nucleotidesubstitutions which do give rise to a different amino acid sequence andtherefore, possibly, a different protein structure which might give riseto an acetyl esterase mutant with different properties than the nativeenzyme. Other examples of possible modifications are insertion of one ormore nucleotides into the sequence, addition of one or more nucleotidesat either end of the sequence, or deletion of one or more nucleotides ateither end or within the sequence.

In still further aspects, the present invention relates to an enzymepreparation useful for the degradation of plant cell wall components,said preparation being enriched in an enzyme with acetyl esteraseactivity as described above, and to various uses of the enzyme or enzymepreparation.

DETAILED DESCRIPTION OF THE INVENTION

According to one embodiment, the enzyme of the invention comprising theconsensus amino acid sequence shown in SEQ ID No. 1, is one whichcomprises the N-terminal amino acid sequence shown in SEQ ID No. 2. Thisenzyme is termed acetyl esterase I in the following disclosure.

In the present context the term "N-terminal amino acid sequence" isintended to be understood in its conventional meaning, i.e. as theN-terminal amino acid sequence of the mature (secreted and processed)enzyme, i.e. the N-terminal sequence remaining after any signal sequenceor pro-sequence have been cleaved off.

Acetyl esterase I of the invention is preferably encoded by a DNAsequence comprising the DNA sequence shown in SEQ ID No. 4 or ananalogue of said DNA sequence, which is at least 70% homologous,preferably at least 80%, and more preferably at least 90% homologouswith said DNA sequence.

Preferably, acetyl esterase I has a molecular weight of about 44 kDa, apI in the range of 4.4-5.1 and/or a pH optimum in the range of about6.0-9.0, such as in the range of about 7.0-7.5 when determined under theconditions described herein.

Acetyl esterase I of the invention has been found to be substantiallydevoid of activity towards acetylated xylan, acetylated mannan and/oracetylated rhamnogalacturonan (as determined by the assays described inthe Materials and Methods section herein). Furthermore, acetyl esteraseI have been found to be devoid of lipase activity.

Another enzyme of the present invention is one, which comprises theN-terminal amino acid sequence shown in SEQ ID No. 3, in which x may beany amino acid residue. This enzyme is termed acetyl esterase II in thefollowing disclosure.

Acetyl esterase II of the invention has been found to be active towardsacetylated xylan or acetylated mannan, and substantially devoid ofactivity towards acetylated rhamnogalacturonan. In fact, acetyl esteraseII of the invention is believed to be the first disclosed enzyme havingactivity towards both acetylated xylan and acetylated mannan.

At present, it is preferred that acetyl esterase II of the invention isone, in which x of the amino acid sequence shown above is a threonineresidue.

Acetyl esterase II of the present invention preferably has a pI in therange of 4-5, such as about 4.5, and/or a molecular weight of about 35kDa.

It will be understood that homologues of the above identified enzymesare to be considered to be within the present invention. In the presentcontext the term "homologue" is intended to indicate an enzyme withacetyl esterase activity which has an N-terminal amino acid sequencediffering in one or more amino acid residues from the sequence shown inSEQ ID No. 2 or 3, respectively, without substantially impairing thecharacteristic acetyl esterase activity of the enzyme. For instance, thehomologue may be a naturally occurring or genetically engineered variantof acetyl esterase I or II of the invention, e.g. prepared by suitablymodifying the DNA sequence encoding the amino acid sequence, resultingin the addition of one or more amino acid residues to either or both theN- and C-terminal end of the sequence, substitution of one or more aminoacid residues at one or more different sites in the amino acid sequence,deletion of one or more amino acid residues at either or both ends of orat one or more sites in the amino acid sequence, or insertion of one ormore amino acid residues at one or more sites in the amino acidsequence.

Preferably, the N-terminal amino acid sequence of the homologous enzymeis at least 70% homologous, such as at least 80%, 90% or 95% homologouswith the N-terminal amino acid sequence shown in SEQ ID No. 2 or 3,respectively.

By the present invention it is possible to provide the acetyl esterasein a highly purified form, i.e. greater than 80% pure, and morepreferably greater than 90% pure as determined by SDS gelelectrophoresis as described in the Materials and Methods sectionherein.

While it is contemplated that enzymes of the invention with acetylesterase activity may be derivable from any source, including plants andmammals, it is presently preferred that the enzyme is of microbialorigin.

In the present context, the term "microbial origin" is intended toinclude bacteria and fungi (such as filamentous fungi or yeasts). Theterm "derivable" is intended to include that the enzyme may be recoveredfrom any of the origins mentioned or may be encoded by and expressedfrom a DNA sequence isolated from or prepared on the basis of DNA fromthe origin in question.

In particular, the enzyme of the invention may be derivable from afungus, more specifically from a strain of Aspergillus, in particular A.aculeatus or A. niger, a strain of Trichoderma, in particular T.harzianum, T. reesie, or a strain of Fusarium, in particular F.oxysporum, or from a strain of Saccharomyces, in particular S.cerevisiae.

As an example, enzymes of the invention with acetyl esterase activitymay be recovered from a culture of any suitable organism, e.g.Aspergillus aculeatus, by purification methods known in the artinvolving ultrafiltration, column chromatography, gel filtration and thelike, and suitable combinations of any of these treatments. Theprotein-containing fractions obtained by these procedures maysubsequently be assayed for acetyl esterase activity. A more detaileddescription of an entire purification scheme is given in Example 1below.

Although the above outlined procedure may be used for producing enzymesof the present invention, it is presently preferred to employrecombinant DNA techniques for this purpose. Accordingly, it ispreferred that the enzyme of the invention is produced by expressionfrom a DNA sequence encoding the enzyme, which is isolated from a cDNAor genomic library of a suitable organism, e.g. as mentioned above. Apreferred example of a suitable organism is Aspergillus aculeatus, CBS101.43, publicly available from the Centraalbureau voorSchimmelcultures, Delft, NL.

The DNA sequence coding for the enzyme may for instance be isolated byscreening a cDNA library of a suitable organism, e.g. Aspergillusaculeatus, and selecting for clones hybridizing with a DNA probeprepared on the basis of an amino acid sequence of said enzyme, such asthe ones described above. The hybridization may be carried out underconditions known in the art, e.g. the conditions described above inconnection with analogous DNA.

Alternatively, the DNA sequence may be isolated by screening a cDNA orgenomic library of any of the organisms mentioned above, in particular astrain of A. aculeatus, and selecting for clones expressing acetylesterase activity as defined above. However, this latter approach may behampered when the cDNA or genomic library is prepared in cells, whichthemselves express acetyl esterase activity, and thereby give rise tofalse positive screening results. Especially, eukaryotic cells areexpected to express acetyl esterase activity, which, when PNP-acetate isused as a substrate, will result in false positive results.

The appropriate DNA sequence selected by either of the above methods maythen be isolated from the clone by standard procedures.

The DNA sequence may subsequently be inserted into a recombinantexpression vector. This may be any vector which may conveniently besubjected to recombinant DNA procedures, and the choice of vector willoften depend on the host cell into which it is to be introduced. Thus,the vector may be an autonomously replicating vector, i.e. a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g. a plasmid. Alternatively,the vector may be one which, when introduced into a host cell, isintegrated into the host cell genome and replicated together with thechromosome(s) into which it has been integrated.

In the vector, the DNA sequence encoding the acetyl esterase enzymeshould be operably connected to a suitable promoter and terminatorsequence. The promoter may be any DNA sequence which showstranscriptional activity in the host cell of choice and may be derivedfrom genes encoding proteins either homologous or heterologous to thehost cell. The procedures used to ligate the DNA sequences coding forthe acetyl esterase, the promoter and the terminator, respectively, andto insert them into suitable vectors are well known to persons skilledin the art (cf., for instance, Sambrook et al., 1989).

The host cell which is transformed with the DNA sequence encoding theenzyme of the invention is preferably a eukaryotic cell, in particular afungal cell such as a yeast or filamentous fungal cell. In particular,the cell may belong to a species of Aspergillus, most preferablyAspergillus oryzae or Aspergillus niger. Fungal cells may be transformedby a process involving protoplast formation and transformation of theprotoplasts followed by regeneration of the cell wall in a manner knownper se. The use of Aspergillus as a host microorganism is described inEP 238 023 (of Novo Nordisk A/S), the contents of which are herebyincorporated by reference. The host cell may also be a yeast cell, e.g.a strain of Saccharomyces, in particular Saccharomyces cerevisiae.

In a still further aspect, the present invention relates to a method ofproducing an enzyme according to the invention, wherein a suitable hostcell transformed with a DNA sequence encoding the enzyme is culturedunder conditions permitting the production of the enzyme, and theresulting enzyme is recovered from the culture.

The medium used to culture the transformed host cells may be anyconventional medium suitable for growing the host cells in question. Theexpressed acetyl esterase may conveniently be secreted into the culturemedium and may be recovered therefrom by well-known procedures includingseparating the cells from the medium by centrifugation or filtration,precipitating proteinaceous components of the medium by means of a saltsuch as ammonium sulphate, followed by chromatographic procedures suchas ion exchange chromatography, affinity chromatography, or the like.

In addition to being used in the preparation of an acetyl esterase ofthe invention, a DNA sequence encoding such acetyl esterase may be usedfor inactivating or destroying genes encoding acetyl esterases. Thus,for some applications, it may be important that acetyl groups arepresent on the polysaccharide substrate to be used, rather than beingremoved by the action of an acetyl esterase. This may be the case forsome applications where a gum is to be used as substrate. Accordingly,the action of acetyl esterases on such substrates may be undesirable.

It is therefore contemplated that a DNA sequence encoding an acetylesterase of the invention (such as the DNA sequence shown in SEQ ID No.4) or a part thereof may be used to avoid the presence of acetylesterase activity in polysaccharide degrading enzyme preparations to beused for applications, in which the presence of acetyl groups on thesubstrate are desirable. More specifically, it is contemplated that aDNA sequence encoding an acetyl esterase of the invention may be usedfor disrupting or inactivating an acetyl esterase gene present andactive in an organism which is to be used as a producer ofpolysaccharide degrading enzymes, substantially free from or comprisingonly a minor amount of an acetyl esterase enzyme.

The inactivation may conveniently be accomplished by preparing anantisense sequence of the acetyl esterase gene and using this antisensesequence for inactivating the acetyl esterase gene(s) in question inaccordance with well-known antisense technology. Hereby, novel enzymeproducing organisms, such as microorganisms, may be produced which arecapable of producing polysaccharide degrading enzymes with a minoramount of (or substantially without) an undesirable acetyl esteraseactivity.

In a still further aspect, the present invention relates to an enzymepreparation useful for the degradation or modification of plant cellwall components, said preparation being enriched in an enzyme withacetyl esterase activity as described above.

The enzyme preparation having been enriched with an enzyme of theinvention may e.g. be an enzyme preparation comprising multipleenzymatic activities, in particular an enzyme preparation comprisingmultiple plant cell wall degrading enzymes such as Pulpzyme, Gamanase,Pectinex®, Pectinex Ultra SP®, Celluclast or Celluzyme (all availablefrom Novo Nordisk A/S). In the present context, the term "enriched" isintended to indicate that the acetyl esterase activity of the enzymepreparation has been increased, e.g. with an enrichment factor of atleast 1.1, conveniently due to addition of an enzyme of the inventionprepared by the method described above.

Alternatively, the enzyme preparation enriched in an enzyme with acetylesterase activity may be one which comprises an enzyme of the inventionas the major enzymatic component, e.g. a mono-component enzymepreparation.

The enzyme preparation may be prepared in accordance with methods knownin the art and may be in the form of a liquid or a dry preparation. Forinstance, the enzyme preparation may be in the form of a granulate or amicrogranulate. The enzyme to be included in the preparation may bestabilized in accordance with methods known in the art.

The enzyme preparation of the invention may, in addition to an acetylesterase of the invention, contain one or more other plant cell walldegrading enzymes, for instance those with cellulytic, xylanolytic,mannanolytic or pectinolytic activities such as α-arabinosidase,xylanase, α-glucoronisidase, β-xylosidase, mannanase, β-mannosidase,α-galactosidase, arabinanase, rhamnogalacturonase, galactanase,polygalacturonase, pectin lyase, pectate lyase, glucanase,α-galacturonisidase, or pectin methylesterase.

The additional enzyme(s) may be producible by means of a microorganismbelonging to the genus Aspergillus, preferably Aspergillus niger,Aspergillus aculeatus, Aspergillus awamori or Aspergillus oryzae, orTrichoderma.

The enzyme preparation according to the invention is preferably used asan agent for degradation or modification of plant cell wall materialsand any xylan or mannan-containing material originating from plant cellwalls.

Examples are given below of preferred uses of the enzyme preparation ofthe invention. The dosage of the enzyme preparation of the invention andother conditions under which the preparation is used may be determinedon the basis of methods known in the art.

Many polysaccharides are known to be acetylated, e.g. acetylated xylansfrom hardwood, softwood or cereals, acetylated galactomannans andgalactoglucomannans from softwood, acetylated pectin from e.g. sugarbeets, acetylated rhamnogalacturonan and acetylated xyloglucan fromdicotyledons, and acetylated gums like gum karaya (from sterculia tree)and is xanthan gum (bacterial gum).

The acetylation of carbohydrates often hinders (fully or partially) thedegradation of the carbohydrate by endoacting enzymes such as thedegradation of acetylated xylan with xylanases or the degradation ofacetylated galactomannan with mannanases.

Accordingly, it is advantageous to use an acetyl esterase of theinvention in combination with xylanases without other xylanolyticenzymes (especially β-xylosidases) or with limited activity of otherxylanolytic enzymes to degrade xylans to oligosaccharides. Sucholigosaccharides may be used as bulking agents.

The degradation of xylan by an acetyl esterase of the invention incombination with a xylanase containing enzyme preparation is facilitatedby full or partial removal of the sidebranches of xylan. Arabinosesidegroups can be removed by a mild acid treatment or byalpha-arabinosidases and the glucuronic acid sidebranches can be removedby alpha-glucuronisidases.

The oligomers released by the combined action of an acetyl esterase anda xylanase, or of an acetyl esterase, a xylanase and asidebranch-hydrolysing enzyme as mentioned above can be further degradedto free xylose (and other monosaccharides) by beta-xylosidases. Thereleased xylose may be converted to other compounds like furanoneflavours.

The acetyl esterase of the present invention can be used in combinationwith a mannanase without other mannanolytic enzymes or with limitedactivity of other mannanolytic enzymes to degrade mannans (includinggalactomannans, glucomannans, and galactoglucomannans) for production ofoligosaccharides. The oligosaccharides may be used as bulking agents.The degradation of mannan by an acetyl esterase of the invention and amannanase containing enzyme preparation is facilitated by full orpartial removal of mannan sidebranches. For instance, the galactosesidebranches of mannan may be removed by alpha-galactosidase.

The oligomers with are released by the acetyl esterase and mannanase orby a combination of acetyl esterase, mannanase and alpha-galactosidaseas mentioned above can be further degraded to free mannose bybeta-mannosidase (and beta-glucosidase for glucomannans andgalactoglucomannans).

Acetyl esterases of the present invention may, in combination withxylanolytic enzymes, be used for modification of animal feed and mayexert their effect either in vitro (by modifying components of the feed)or in vivo. The acetyl esterases are particularly suited for addition toanimal feed compositions containing high amounts of acetylated xylans,e.g. feed containing grass, or leaves of cereals or maize.

Acetyl esterases of the present invention may in combination withxylanases and/or mannanases be used in the paper and pulp industry, e.g.to improve the bleachability or drainability of lignocellulosic pulp.

Plant material may be treated with an acetyl esterase of the inventionin combination with other enzymes in order to improve different kinds ofprocessing, facilitate purification or extraction of different componentlike carbohydrates, improve the feed value, decrease the water bindingcapacity, improve the degradability in waste water plants, improve theconversion of e.g. grass and corn to ensilage, or to hydrolyse variousplant cell wall-derived materials or waste materials, e.g. from paperproduction, or agricultural residues such as wheat-straw, corn cobs,whole corn plants, nut shells, grass, vegetable hulls, bean hulls, spentgrains, sugar beet pulp, and the like.

The acetyl esterase preparation of the invention may be used todeacetylate carbohydrates in order to change properties thereof, such asthe rheology, the stabilizing ability or the hydrophobicity of thecarbohydrates. Examples are deacetylation of xylans and mannans. Thecarbohydrate acetyl esterase preparation to be used for the abovepurpose is preferable essentially free from activities with candepolymerize said carbohydrates.

Furthermore, in systems with low water activity acetyl esterases of theinvention may be used to esterify, e.g. acetylate (by ester synthesis ortransesterification), carbohydrates like xylans and mannans. The therebyformed acetylated carbohydrates may be more acetylated than naturallyoccurring carbohydrates, and will have new properties like increasedhydrophobicity and thereby improved ability to emulsify and/or stabilizefat-containing emulsions.

The enzyme preparation of the invention may be used for degrading plantcell wall polysaccharides in the food industry, primarily in fruit andvegetable processing such as fruit juice production or wine making, andin the paper and pulp industry. Furthermore, the enzyme preparation maybe used for modification or degradation of gums, e.g. used in foodapplications. Examples of such gums are gum Karaya (from sterculiatree), gum arabic, and in particular Locust Bean Gum (carob tree), guargum and xanthan gum (bacterial gum).

The invention is further illustrated in the drawing, in which

FIG. 1 is a flowsheet illustrating the purification procedure used forisolating enzymes of the invention,

FIG. 2 a silver stained SDS-PAGE gel of purified acetylesterasesisolated from the A.aculeatus supernatant described herein. The proteinswere separated on a 12% T SDS-PAGE gel and silverstained as described inthe materials and methods section herein. Lane 1: molecular weightmarkers (Phosphorylase B: 94 kDa, BSA: 67 kDa, Ovalbumin: 43 kDa,Carbonic anhydrase: 30 kDa, STI: 20.1 kDa). Lane 2: 10 μg of A.aculeatus supernatant. Lane 3: 20 μg Pool I. Lane 4: 10 μg of aside-fraction obtained from anion exchange purification. Lane 5: 0.8 μgPool I.I. Lane 6: 0.07 μg Pool I.II. Lane 7: 1.2 μg purifiedrhamnogalacturonan acetyl esterase (WO 92/19728);

FIG. 3 illustrates the. pH optimum of the acetyl esterase recovered frompool I.I determined as described in the materials and methods sectionbelow;

FIG. 4 the pH optimum of the acetyl esterase recovered from pool I.II asdetermined on PNP acetate, acetylated mannan and acetylated xylan; and

FIG. 5 the iso-electric point of an enzyme of the invention determinedby isoelectric focusing. Lane 1: isoelectric point of markerAmyloglycosidase (pI 3.5), trypsin inhibitor (pI 4.55), bovine carbonicanhydrase (pI 5.85), human carbonic anhydrase (pI 6.55). Lane 2: 10 μgA. aculeatus culture supernatant. Lane 3: 1 μg Pool I.I. Lane 4: 1 μgPool I.II.

The invention is described in further detail in the following exampleswhich are not in any way intended to limit the scope of the invention asclaimed.

MATERIALS AND METHODS

Donor organism and fermentation: Aspergillus aculeatus, strain CBS101.43, was grown in a medium containing 12% w/w of soy-cake and 1.5%potassium phosphate with a pH lower than 5.0 in a 40 m³ aeratedcontinuously stirred fermenter. The pH was kept at approx. 4.2 duringthe fermentation, and potato starch was added continuously during thefermentation. The resulting culture supernatant was filtered,centrifugated and concentrated in a 20 kDa UF filter to approx. 100mg/ml protein.

PNP-acetyl esterase activity: The activity was measured by incubating asample of the purified enzyme in 500 μl of 1.6 mM PNP-acetate (Sigma,St. Louis, U.S.A.); 20 mM citrate pH 5.5 and quantifying the amount ofPNP released by measuring OD₄₀₅ after addition of 100 μl 1M Tris, pH7.0. One unit is defined as the activity which releases 1 μmole PNP/min.at 40° C.

Acetyl esterase assay (Boehringer Mannheim assay): The assay isperformed as described in the manufacturers manual.

The substrate solutions to be used in the assay are prepared bydissolving the acetylated polysaccharide substrate to a 1%solution/suspension in reagent grade water.

65 μl of the substrate solution is added to 30 μl of 0.3M buffer of anappropriate pH (i.e. Tris buffer pH 6.5 for Pool I.I and I.II acetylesterases). Finally 5 μl enzyme solution is added (as well as blankswith 5 μl water).

The samples are incubated 1 hour at 37° C. on a shaking table and thenheated to 95° C. for 15 min. to inactivate the enzyme.

10 μl of this solution is used to measure acetate content by theBoehringer Manheim assay.

Acetylated substrates:

Acetylated Rhamnogalacturonan (Modified Hairy Regions)

The substrate was obtained from Golden Delicious Apples by partialpectinolytic degradation with a pectinase preparation (Pectinex AR,Novo-Nordisk), followed by centrifugation, aroma-stripping andultrafiltration on a MW 60,000 Da. cut-off BX3 polysulfone membrane(Paterson Candy Ltd.) The retentate was dialysed against water andlyophilized. The MHR fraction represents 1,7% of the apple solids andapproximately 4% of the saccharides are acetylated.

Acetylated Mannan

The substrate was kindly provided by Jurgen Puls (Hamburg, FRG). It wasisolated from Norwegian Bruce (Picia abes) by delignifying sawdust withchlorite according to standard procedures, followed by DMSO extractionof hemicellulose. Extracted polysaccharides were separated by anionexchange on a DEAE sepharose column, and the fractions containing mannanwere collected.

Acetylated Xylan

Acetylated xylan was obtained as a non-dialysable fraction of watersoluble polysaccharide produced by steam extraction of birchwood asdescribed in reference 6 (Kormelink & Voragen). It contained 70.6% totalsugars and 10.6% acetyl (based on dry matter).

SDS-page electrophoresis was performed in a Mini-Leak 4 electrophoresisunit (Kem-En-Tec, Denmark) as a modified version of the Laemli procedure16!. Isoelectric focusing was carried out on Ampholine PAG plates pH3.5-9.5 (Pharmacia, Sweden) on a Multiphor electrophoresis unitaccording to the manufactures instructions. Gels were eithersilverstained essentially as described in 11! or coomassie stained.

Determination of pI: The isoelectric points were determined byelectrophoresis on a Pharmacia Ampholine PAG plate (pH 3.5-9.5)according to the manufacturers instructions. After electrophoresis thegel was silver stained as described below.

Protein assay: The BioRad protein assay was used.

Determination of pH optimum:

with PNP-acetate

The purified enzyme (2-5 μg) was incubated with 2 mM PNP-acetate in 50mM citrate-phosphate buffer of various pH. After 25 min incubation, thePNP release was quantified by measuring OD_(405nm) in 0.2M Tris pH 7.0.Control samples containing PNP-acetate and buffer, but no enzyme wereincluded and the absorbance measured in these samples were subtractedfrom the enzyme containing samples.

with acetylated polysaccharides

For determination of activity of the enzyme on acetylatedpolysaccharides, the sample was incubated with a 1% substrate solutionin 0.1M citrate/phosphate buffer of various pH, and after 30 minincubation, the released acetate was measured by the Boehringer Mannheimassay. Control samples without enzyme were included, and subtracted fromthe enzyme containing samples.

Amino acid sequencing: The purified enzymes were run on an SDS-PAGE asdescribed above and electro-blotted to a PVDF-membrane (Immobillon,Milipore, U.S.A.) using standard procedures. The electro-blottedproteins were subjected to N-terminal amino acid sequence determinationon an Applied Biosystems 473A sequencer operated in accordance with themanufacturers instructions.

mRNA preparation: mRNA may be isolated from Aspergillus aculeatus, CBS101.43, grown as described above, by harvesting mycelia after 3-5 days'growth, immediately freezing in liquid nitrogen and storing at -80° C.

Construction of an A. aculeatus cDNA library in E. coli: was performedsubstantially as described in WO 92/19728, the contents of which ishereby incorporated by reference. More specifically, total RNA isextracted from homogenized A. aculeatus mycelium using methods asdescribed by Boel et al. (EMBO J., 3: 1097-1102, 1984) and Chirgwin etal. (Biochemistry (Wash), 18: 5294-5299, 1979). Poly(A)-containing RNAis obtained by two cycles of affinity chromatography onoligo(dT)-cellulose as described by Aviv and Leder (PNAS, USA69:1408-1412, 1972). cDNA is synthesized with the use of a cDNAsynthesis kit from Invitrogen according to the manufacturer'sdescription.

Identification of A. aculeatus acetyl esterase specific cDNArecombinants: was performed by use of syntheticoligodeoxyribonucleotides prepared on the basis of the determined aminoacid sequences of the acetyl esterases or by use of immunologicalscreening procedures. These procedures are described in detail in WO92/19728.

Construction of an Asperaillus expression vector: may be performed byinserting a DNA sequence obtained as described above in the expressionvector pHD414 described in WO 92/19728. The resulting expression plasmidmay be amplified in E. coli in accordance with well-known procedures,and subsequent transformed into A. oryzae or A. niger according to thegeneral procedure described below.

Transformation of Aspergillus oryzae or Aspergillus niger (generalprocedure)

100 ml of YPD (Sherman et al., Methods in Yeast Genetics, Cold SpringHarbor Laboratory, 1981) is inoculated with spores of A. oryzae or A.niger and incubated with shaking at 37° C. for about 2 days. Themycelium is harvested by filtration through miracloth and washed with200 ml of 0.6M MgSO₄. The mycelium is suspended in 15 ml of 1.2M MgSO₄.10 mM NaH₂ PO₄, pH=5.8. The suspension is cooled on ice and 1 ml ofbuffer containing 120 mg of Novozym® 234, batch 1687 is added. After 5minutes 1 ml of 12 mg/ml BSA (Sigma type H25) is added and incubationwith gentle agitation continued for 1.5-2.5 hours at 37° C. until alarge number of protoplasts is visible in a sample inspected under themicroscope.

The suspension is filtered through miracloth, the filtrate transferredto a sterile tube and overlayered with 5 ml of 0.6M sorbitol, 100 mMTris-HCl, pH=7.0. Centrifugation is performed for 15 minutes at 100 gand the protoplasts are collected from the top of the MgSO₄ cushion. 2volumes of STC (1.2M sorbitol, 10 mM Tris-HCl, pH=7.5. 10 mM CaCl₂) areadded to the protoplast suspension and the mixture is centrifugated for5 minutes at 1000 g. The protoplast pellet is resuspended in 3 ml of STCand repelleted. This is repeated. Finally the protoplasts areresuspended in 0.2-1 ml of STC.

100 μl of protoplast suspension is mixed with 5-25 μg of the appropriateDNA in 10 μl of STC. Protoplasts are mixed with p3SR2 (an A. nidulansamdS gene carrying plasmid). The mixture is left at room temperature for25 minutes. 0.2 ml of 60% PEG 4000 (BDH 29576). 10 mM CaCl₂ and 10 mMTris-HCl, pH=7.5 is added and carefully mixed (twice) and finally 0.85ml of the same solution is added and carefully mixed. The mixture isleft at room temperature for 25 minutes, spun at 2500 g for 15 minutesand the pellet is resuspended in 2 ml of 1.2M sorbitol. After one moresedimentation the protoplasts are spread on the appropriate plates.Protoplasts are spread on minimal plates (Cove Biochem.Biophys.Acta 113(1966) 51-56) containing 1.0M sucrose, pH=7.0, 10 mM acetamide asnitrogen source and 20 mM CsCl to inhibit background growth. Afterincubation for 4-7 days at 37° C. spores are picked and spread forsingle colonies. This procedure is repeated and spores of a singlecolony after the second reisolation is stored as a defined transformant.

Immunological cross-reactivity: Antibodies to be used in determiningimmunological cross-reactivity may be prepared by use of a purifiedacetyl esterase. More specifically, antiserum against an enzyme of theinvention may be raised by immunizing rabbits (or other rodents)according to the procedure described by N. Axelsen et al. in: A Manualof quantitative Immunoelectrophoresis, Blackwell ScientificPublications, 1973, Chapter 23, or A. Johnstone and R. Thorpe,Immunochemistry in Practice, Blackwell Scientific Publications, 1982(more specifically pp. 27-31). Purified immunoglobulins may be obtainedfrom the antisera, for example by salt precipitation ((NH₄)₂ SO₄),followed by dialysis and ion exchange chromatography, e.g. onDEAE-Sephadex. Immunochemical characterization of proteins may be doneeither by Outcherlony double-diffusion analysis (O. Ouchterlony in:Handbook of Experimental Immunology (D. M. Weir, Ed.), BlackwellScientific Publications, 1967, pp. 655-706), by crossedimmunoelectrophoresis (N. Axelsen et al., supra, Chapters 3 and 4), orby rocket immunoelectrophoresis (N. Axelsen et al., Chapter 2).

EXAMPLE 1 Purification

The purification of the acetyl esterases was carried out as outlined inFIG. 1.

Initial purification

100 ml of Aspergillus aculeatus supernatant obtained as described abovewere ultrafiltrated in a 200 ml Amicon cell with a 10 kDa membrane. Theretentate was diluted 10 times with 20 mM tris pH 7.0, adjusted to afinal volume of 50 ml, and applied to a 250 ml DEAE column (50 mminternal diameter) in 20 mM tris pH 7.0 at a flow rate of 4 ml/min.Bound proteins were eluted with a linear gradient from 0.1 to 0.4M NaClover 200 min., and 10 ml fractions were collected and assayed forPNP-acetyl-esterase activity. One peak of PNP-acetyl esterase activitywas eluted (Pool I) as indicated in FIG. 1.

To pool I ammonium sulphate (AMS) was added to a final concentration of2M and the resulting mixture was applied to a 60 ml Phenyl sepharosecolumn (26 mm internal diameter) in 50 mM tris pH 6.5; 2M AMS at a flowrate of 2 ml/min. Bound proteins were eluted with a step gradient offalling AMS concentration (a decrease of 0.5M AMS per step), and theDEAE pool was separated into two distinct peaks of activity (Pool I.Iand Pool I.II).

The pools from the phenyl Sepharose column were purified bygelfiltration on a 500 ml Superdex 6.75 column (26 mm internal diameter)in 0.25M ammonium acetate, pH 5.5, at a flow rate of 1 ml/min. 10 mlfractions were collected and assayed for PNP-acetyl-esterase activityusing the above described procedure.

Final Purification of Partially Purified Acetyl Esterase ContainingFractions

Pool I.I (acetyl esterase I):

This pool of activity was ultrafiltrated into 10 mM sodium phosphate pH6.8, and applied to a 8 ml BioRad HTP hydroxyl apatite column (10 mminternal diameter) at a constant flow rate of 1 ml/min. Bound proteinswere eluted with a linear increasing phosphate concentration to 0.5Mover 30 min. 5 ml fractions were collected and assayed forPNP-acetyl-esterase activity and protein content (using SDS-PAGE andprotein assay). The purified enzyme had a molecular weight of 44 kDa asdetermined by SDS-PAGE as described above. The purified enzyme did notshow any activity towards acetylated xylan, mannan orrhamnogalacturonan.

Pool I.II (acetyl esterase II)

After gelfiltration, a fraction, which has activity on both acetylatedmannan and xylan, was obtained. This fraction was ultrafiltrated into 10mM sodium phosphate pH 6.8 and applied to a 8 ml BioRad HTP hydroxylapatite column (10 mm internal diameter) at a constant flow rate of 1ml/min. Bound proteins were eluted with a linear increasing phosphateconcentration to 0.5M over 30 min. 5 ml fractions were collected andassayed for PNP-acetyl-esterase activity and protein content (SDS-PAGEand protein assay). The activity was recovered in two fractions, both ofwhich contain a 42 kDa protein and a 35 kDa protein. The 42 kDa proteinis assumed to be the polygalacturonase I described in PCT/DK93/00445.The last eluted fraction was ultrafiltrated into 25 mM tris pH 8.0 andis applied on a 1 ml mono-P column at 1 ml/min. Bound proteins wereelluted with a linear increasing NaCl gradient from 0 to 0.5M NaClduring 60 min. 2 ml fractions were collected and assayed forPNP-acetyl-esterase activity and protein content (SDS-PAGE and proteinassay). The fraction with activity contains one 35 kDa protein which isactive on both acetylated mannan and xylan.

In the following table, purification factors and activity of A.aculeatus acetyl esterases obtained during the above describedpurification procedure are listed. Selected fractions from thepurification procedure are shown, as well as the final purified enzymefractions. The recovery of PNP-acetyl esterase activity was 0.8%. Theacetyl esterase activity was measured by the procedure given above.

    ______________________________________                                                Protein  Activity   Specific                                                                              Purification                              Fraction                                                                              content  (μmol/min)                                                                            activity                                                                              factor                                    ______________________________________                                        A.a. sup.*.sup.)                                                                      10     g     6320.00 U                                                                              0.632 U/mg                                                                            1.0                                     Pool I  950    mg    2148.00 U                                                                              2.26 U/mg                                                                             3.6                                     Pool I.I                                                                              21     mg    345.00 U 16.87 U/mg                                                                            26.7                                    after S-200                                                                   Pool I.II                                                                             192    mg    574.20 U 2.47 U/mg                                                                             3.9                                     after S-200                                                                   Pool I.I                                                                              200    μg 3.49 U   17.46 U/mg                                                                            27.6                                    Pool I.II                                                                             200    μg 20.00 U  100.00 U/mg                                                                           158.2                                   ______________________________________                                         *.sup.) A. aculeatus supernatant                                         

EXAMPLE 2 Characterization of Purified Acetyl Esterases of the Invention

The pH optimum of the purified acetyl esterases described in Example 1above was determined using the procedures described in the Materials andMethods section above.

The acetyl esterase isolated from Pool I.I (and having an apparentmolecular weight of 44 kDa) was found to have a pH optimum of 7.0-7.5when using PNP-acetate as a substrate. The enzyme was found to be activefrom pH 5.0 to pH 9.0. The pH profile is shown in FIG. 3.

The acetyl esterase isolated from Pool I.II was found to have a minor pHoptimum of 6.0 and a major pH optimum of pH 7-9 using PNP-acetate as asubstrate. However, this high activity at alkaline pH may be an artefactcaused by the instability of the substrate above neutral pH. Usingacetylated mannan and xylan as substrate a broad pH optimum with a peakat 6.0 was detected. The pH profile is shown in FIG. 4.

The isoelectric points of the two enzymes were determined by use of theprocedure described in the Materials and Methods section above. Theresults are shown in FIG. 5.

The N-terminal amino acid sequence of the two enzymes were determinedusing the procedure described in the materials and methods sectionabove.

EXAMPLE 3 Generation of a cDNA Probe by Polymerase Chain Reaction

To obtain a cDNA probe for an acetyl esterase from A. aculeatus, adegenerate oligonucleotide (primer AE 1.1/p3;p3γ-5'-TTYGAYTGGGAYWSIAC-3') (SEQ ID NO:6) corresponding to a region inthe NH₂ -terminal sequence of the purified acetyl esterase I wassynthesized by incorporating deoxyinosines at the ambiguous position.The primer was used pairwise with the direct (primer #22,5'-CTGTAATACGACTCACTA-3') (SEQ ID NO:7) and reverse (primer #1725'-GGGCGTGAATGTAAGCGTGAC-3') (SEQ ID NO:8) pYES 2.0 primers to amplifythe target acetyl esterase cDNA from an amplified cDNA library poolcontaining 7000 clones employing the polymerase chain reaction technique(Ohara et al. 1989). The PCR reactions were carried out in 100 μl PCRbuffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.01%,Perkin-Elmer, Cetus) containing 550 pmol of sense primer (AE1.1/p3) and100 pmol of each antisense primer (see above), 1 μg template DNA(Qiagen-purified plasmid DNA) and 200 μM each dNTP using a DNA thermalcycler and 2.5 units of Taq polymerase (Perkin-Elmer, Cetus). Thirtycycles of PCR were performed using a cycle profile of denaturation at94° C. for 1 minute, annealing at 55° C. for 2 minutes, and extension at72° C. for 3 minutes.

Ten μl aliquots of the amplification products were analyzed byelectrophoresis on 1% agarose gels revealing a 1.5 kb and a 1.0 kbproduct with one primer pair (AE1.1/p3; pYES reverse primer #172). TheDNA fragment of interest was excised from the gel, recovered byelectroelution (Sambrook et al. 1989) using type D-0405 seamlessdialysis tubing (Sigma), followed by phenol extraction and ethanolprecipitation at -20° C. for 12 hours. The PCR product was blunt-endedat 37° C. for 10 minutes in 20 μl buffer (20 mM Tris-acetate, pH 7.9, 10mM MgAc, 50 mM KAc, 1 mM DTT) containing 50 μM each dNTP and 3 units ofT4 DNA polymerase (New England Biolabs). The reaction was stopped byincubation at 70° C. for 5 minutes, chilled on ice for 5 minutes anddiluted in 50 μl of kinase buffer (70 mM Tris-HCl, pH 7.6, 10 MM MgCl₂,5 mM DTT) followed by phosphorylation at 37° C. for 30 minutes with T4polynucleotide kinase (10 U, New England Biolabs) and 1 mM ATP pH 7.0(Pharmacia), phenol extraction, ethanol precipitation and ligation at16° C. for 12 hours into Sma I-cut, dephosphorylated pUC18 vector (50 ngper ligation, Pharmacia).

E. coli DH5α was transformed to ampicillin resistance (Hanahan 1985)using 5 μl of the ligation mixture, and 10 clones were analyzed byisolation of plasmid miniprep. DNA (Sambrook et al. 1989), andEcoRI/HindIII digestion of the plasmid subclones, followed by sequencingthe ends of the 1.5 kb insert from one subclone (pAE 8.1.1) withuniversal pUC primers (Sanger et al. 1977). Nucleotide sequence analysisof the pC1AE1 subclone revealed a unique open reading frame which, inaddition to the primer encoded amino acids, contained additionalresidues concurring with the available NH₂ -terminal sequence from thepurified acetyl esterase I.

Isolation and Characterization of a Full-length cDNA Encoding an AcetylEsterase from A. aculeatus

To isolate a full-length cDNA clone for an acetyl esterase, coloniesfrom the cDNA library pool #22 were plated on LB-agar (24×24 cm plate,Nunc) containing ampicillin (100 μg/ml) and replicated on anLB+amp-plate covered with a nylon filter (Hybond-N, Amersham). Thepurified 1.5 kb acetyl esterase PCR fragment was ³² P-labeled byrandom-priming as above, and used as a probe in screening the librarypool by colony hybridization (Sambrook et al., 1989). The hybridizationwas carried out in 2×SSC (Sambrook et al. ,1989), 5×Denhardt's solution(Sambrook et al. 1989), 1% (W/V) SDS and 100 μg/ml denatured salmonsperm DNA with a probe concentration of 2.5 ng/ml for 16 hours at 65°C., then in 2×SSC (2×15 minutes), 0.2×SSC, 1% SDS (2×30 minutes), and in2×SSC (2×15 minutes) followed by autoradiography at -80° C. for 12hours. Screening of 10,000 colonies from pool 22 yielded 10 putativeacetyl esterase I cDNA clones that were colony-purified by two morerounds of hybridizations. One of these clones (designated pC1AE1) wascharacterized by digesting the plasmid with HindIII and XbaI andsequencing the ends of the 1.4 kb cDNA insert with forward and reversepYES 2.0 polylinker primers.

The 1.4 kb insert in pC1AE1 contains a 1019 bp open reading frame (ORF)initiating with an ATG codon at nucleotide position 33 and terminatingwith a TGA stop codon (SEQ ID No. 4). SEQ ID No. 4 shows the nucleotidesequence of the 3'-end of the acetyl esterase cDNA and SEQ ID No. 5 thededuced primary structure of an acetyl esterase from A. aculeatuscontemplated to be highly homologous to acetyl esterase I. The ORF ispreceded by a 39 bp 5' non-coding region and followed by a 132 bp 3'non-coding region and a poly(A) tail. Comparison of the deduced proteinsequence with the NH₂ -terminal sequence of the purified mature acetylesterase I reveals that the cDNA encodes a precursor protein containinga 27 residue signal peptide and possibly propeptide.

EXAMPLE 4

In order to express the enzyme in Aspergillus, cDNA is isolated from oneor more representatives of each family by digestion with HindIII/XbaI orother appropriate restriction enzymes, size fractionation on a gel andpurification and subsequently ligated to pHD414. After amplification inE. coli, the plasmids are transformed into A. oryzae or A. nigeraccording to the general procedure described above.

REFERENCES CITED IN THE SPECIFICATION

1. Carpita, N. C. and D. M. Gibeaut, (1993), "Structural models ofprimary cell walls in flowering plants: consistency of molecularstructure with the physical properties of the walls during growth", ThePlant Journal 3(1), 1-30,

2. Sutherland, I. W., (1992), "The role of acylation inexopolysaccharides including those for food use", Food Biotechnology,6(1), 75-86,

3. McKay, A. M., (1993), "Microbial carboxylic ester hydrolases (EC3.1.1) in food biotechnology", Letters in Applied Microbiology, 16, 1-6,

4. Dekker, R. F. and G. N. Richards, (1976), "Hemicellulases: Theiroccurence, purification, properties and mode of action", in Advances incarbohydrate chemistry and biochemistry, R. S. Tipson and D. Horton,Editor, Academic Press: New York. p. 277-352,

5. Ward, O. P. and M. Moo-Young, (1989), "Enzymatic Degradation of CellWall and Related Plant Polysaccharides". CRC Critical Reviews inBiotechnology, 8(4): p. 237-274,

6. Searle-van Leeuwen, M. J. F., et al., (1992), "Rhamnogalacturonanacetyl esterase: a novel enzyme from Aspergillus aculeatus, specific forthe deacetylation of hairy (ramified) regions of pectin". Appl.Microbiol. Biotechnol., 38: p. 347-349,

7. Kormelink, F. J. M., et al., (1992), "The purification andcharacterization of an acetyl xylan esterase from Aspergillus niger". J.Biotechnol., 27 267-282,

8. Khan, A. W., K. A. Lamb, and R. P. Overend, (1990), "Comparison ofnatural hemicellulose and chemically acetylated xylan as substrates forthe determination of acetyl-xylan esterase activity in Aspergilli".Enzyme Microb. Technol., 12: p. 127-131,

9. Sundberg, M. and K. Poutanen, (1991), "Purification and properties oftwo acetylxylan esterases of Tricoderma reesi". Biotechnol. Appl.Biochem., 13: p. 1-11,

10. Biely, P., J. Puls, and H. Schneider (1985), "Acetyl xylan esterasesin fungal cellulytic systems".FEBS Lett., 186: p 80-84,

11. Mose Larsen, P., (1981) "An assessment of the potential offered bytwo-dimensional gel electrophoresis and silverstaining for developmentalbiology". Dissertation, Århus university.

12. Sambrook, J., Fritsch, E. F. & Maniatis, T. 1989. Molecular Cloning:A Laboratory Manual. Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.

13. Sanger, F., Nicklen, S. & Coulson, A. R. 1977. Proc. Natl. Acad.Sci. U.S.A. 74: 5463-5467.

14. Ohara, O., Dorit, R. L. & Gilbert, W. 1989. Proc. Natl. Acad. Sci.U.S.A. 86:5673-5677.

15. Hanahan, D. 1985. in DNA Cloning, (Glover, D. M., ed.) IRL, Oxford,Vol. 1., pp. 109-135.

16. Laemmli, U. K., 1970, "Cleavage of structural proteins during theassembly of the head of bacteriophage T4"., Nature, 227, p. 680-685

17. Aviv, H. & Leder, P. 1972. Proc. Natl. Acad. Sci. U.S.A. 69:1408-1412;

18. Becker, D. M. & Guarante, L. 1991. Methods Enzymol. 194: 182-187;

19. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J.1979. Biochemistry 18: 5294-5299;

20. Gubler, U. & Hoffman, B. J. 1983. Gene 25: 263-269.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      IleXaaPheGlyAspXaaTyrTyrThr                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      PheAspTrpAspSerThrLysTyrLeuLeuIleAlaPheGlyAspSer                              151015                                                                        TyrTyrThrValGln                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      ThrThrLysTyrValIleSerPheGlyAspAspTyrTyrThrThrXaa                              151015                                                                        Phe                                                                           (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1315 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      GAATTCCAGGGACTTCCATTCGAGCCATAGCCATGCTCGCTCTCTGGCAGTGCCTAGCCC60                TCGCGGCCATCCCTACAGTATCAGCATTCCCCTCCGGTTCGTCTCAGAAGGCGTTCGACT120               GGGACTCGACGAAATACCTAATAGCGTTCGGTGATTCCTATACGTACGTGCAGGGCACTC180               ACGGACACCAAAACTATAGCTTCATTGGGGATCTGCAGAACTTCGCATATGATGCTCAGA240               CCCTGTTAACGGATAAGATCGTTCAGAACCAGACAGCAACGGCAGAAGGGGGCCCCAACT300               GGGTTGAATACCTCACCGGGTGCGGGGTAGAAGATGGAATCATCTCACCCTTGGACTGCG360               AGAGACAACTCTGGGACTTCGCGTTCGCGGGATCTGATATCTCCGTTGCATACACCCCCC420               TCCACCACAACTACACCGTCTCCCTCGTCAACCAGGTCANCCAATTCACGACCTACGGGC480               AGCCGGTCCTCTCGCGGCACATCTCCGCGCCGCAGACCCTTGTCGCCATCTGGATCGGGA540               TCAACGACATCGGCGACAGCGCCAAATACGCCGTCGATTTTCCGGCCTTCTACGAAACCC600               TCATCACCACCCTCTTCGCCTCCGTCCAGGAGATCTACGCCCAGGGCTATCGCTCCTACC660               TGTTCGTCAACCTGCCGCCCCTCGACCGCACCCCGGNCAACCAGGCCCTGAGCCAGCCCT720               ACCCGAACGCCACGCAGGTCGCCTGGTACAACGACGCGCTGGCCCGGAACGCCGCCGCCT780               TCCACCGCAACCACACGGACACGGCCGTGCACCTGTTNGACGCGCACCGGACGCTCAGCG840               AGGTCATGGACCACCCCGCGGCGTACGGCATCGTCAACACCACCAACTTCTGCCCCGGGT900               ACGACCAGCCCGATATCGCGTGGAACTACCGGGCGTACGGGTGTCCGACCCCGCTGGAGG960               AGTACTTCTGGTTCAACTCGGGGCATCTGACGAGCCATGTGCATCAGATTCTTGCGGGTG1020              TGTTGGAGGGGGAGCTGAGAGAGTGGTCGAAGTGAGGGTGGTCTGTCGTTGATTGGAGGC1080              GTGGTGGGGGGAACTCATTGATGATCCAGTGGGAATACGTCAGCTCCAAACTATGCTTTG1140              TACGCTTCAGTTTAGACTGACGCAGGTAAGACTCCGTAGCATGATTCATCACAACAATCC1200              CGGACCTGCATGCATTTAAGTTGGGTGTATACCACTGGTATCTGCTTGTTACTCCTCGTA1260              TATGTCAACCATATGAGAAGTCAAATATGCCATCGCGTGAAAAAAAAAAAAAAAA1315                   (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 340 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      MetLeuAlaLeuTrpGlnCysLeuAlaLeuAlaAlaIleProThrVal                              151015                                                                        SerAlaPheProSerGlySerSerGlnLysAlaPheAspTrpAspSer                              202530                                                                        ThrLysTyrLeuIleAlaPheGlyAspSerTyrThrTyrValGlnGly                              354045                                                                        ThrHisGlyHisGlnAsnTyrSerPheIleGlyAspLeuGlnAsnPhe                              505560                                                                        AlaTyrAspAlaGlnThrLeuLeuThrAspLysIleValGlnAsnGln                              65707580                                                                      ThrAlaThrAlaGluGlyGlyProAsnTrpValGluTyrLeuThrGly                              859095                                                                        CysGlyValGluAspGlyIleIleSerProLeuAspCysGluArgGln                              100105110                                                                     LeuTrpAspPheAlaPheAlaGlySerAspIleSerValAlaTyrThr                              115120125                                                                     ProLeuHisHisAsnTyrThrValSerLeuValAsnGlnValXaaGln                              130135140                                                                     PheThrThrTyrGlyGlnProValLeuSerArgHisIleSerAlaPro                              145150155160                                                                  GlnThrLeuValAlaIleTrpIleGlyIleAsnAspIleGlyAspSer                              165170175                                                                     AlaLysTyrAlaValAspPheProAlaPheTyrGluThrLeuIleThr                              180185190                                                                     ThrLeuPheAlaSerValGlnGluIleTyrAlaGlnGlyTyrArgSer                              195200205                                                                     TyrLeuPheValAsnLeuProProLeuAspArgThrProXaaAsnGln                              210215220                                                                     AlaLeuSerGlnProTyrProAsnAlaThrGlnValAlaTrpTyrAsn                              225230235240                                                                  AspAlaLeuAlaArgAsnAlaAlaAlaPheHisArgAsnHisThrAsp                              245250255                                                                     ThrAlaValHisLeuXaaAspAlaHisArgThrLeuSerGluValMet                              260265270                                                                     AspHisProAlaAlaTyrGlyIleValAsnThrThrAsnPheCysPro                              275280285                                                                     GlyTyrAspGlnProAspIleAlaTrpAsnTyrArgAlaTyrGlyCys                              290295300                                                                     ProThrProLeuGluGluTyrPheTrpPheAsnSerGlyHisLeuThr                              305310315320                                                                  SerHisValHisGlnIleLeuAlaGlyValLeuGluGlyGluLeuArg                              325330335                                                                     GluTrpSerLys                                                                  340                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TTGATGGGAAC11                                                                 (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CTGTAATACGACTCACTA18                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GGGCGTGAATGTAAGCGTGAC21                                                       __________________________________________________________________________

We claim:
 1. An isolated and purified enzyme with acetyl esteraseactivity obtained from a strain of Aspergillus aculeatus, which enzymeexhibits activity towards acetylated xylan and acetylated mannan andwhich enzyme comprises the amino acid sequence shown in SEQ ID No.1, inwhich Xaa denotes any amino acid residue.
 2. The enzyme according toclaim 1, which enzyme comprises the N-terminal amino acid sequence shownin SEQ ID No. 3, in which Xaa denotes any amino acid residue.
 3. Theenzyme according to claim 2, in which Xaa of the N-terminal amino acidsequence is T.
 4. The enzyme according to claim 1, which has a pI ofabout 4-5.
 5. The enzyme according to claim 1, which has a molecularweight of about 35 kDa.
 6. The enzyme according to claim 1, which enzymeis derivable from Aspergillus aculeatus, CBS 101.43.
 7. The enzymeaccording to claim 1, which is encoded by a DNA sequence isolated from aDNA library of Aspergillus aculeatus, CBS 101.43.
 8. An enzymepreparation useful for the for the treatment of plant cell wallcomponents, said preparation being enriched in an enzyme with acetylesterase activity according to claim
 1. 9. The preparation according toclaim 8, which further comprises a xylanase, a mannase, a pectin lyase,a rhamnogalacturonase, a polygalacturonase, arabinase, galactanase,glucanase, and/or pectin methylesterase.
 10. A method for degrading ormodifying plant material comprising treating said plant material with anamount of the preparation of claim 9 effective to modify or degrade saidplant material.
 11. The method according to claim 10, in whichacetylated xylan and/or acetylated mannan is degraded or modified insaid plant material.